This invention relates to mouse and human Nramp cDNAs (natural resistance-associated macrophage protein), responsible for the natural resistance to infection with intracellular parasites, and to the isolation of Nramp sequences from other animal sources.
The cDNAs can be expressed in expression constructs. These expression constructs and the proteins produced therefrom can be used for a variety of purposes including diagnostic and therapeutic methods.
The ability of a host to resist infection with a wide range of viral, bacterial, and parasitic pathogens is strongly influenced by genetic factors (reviewed by Skamene, 1985, Prog. Leukoc. Biol. 3: 111-559; Skamene and Pietrangeli, 1991, Curr. Opin. Immunol. 3, 511-517). A clear manifestation of differential resistance or susceptibility of human populations in endemic areas of disease and during epidemics has been observed in the case of mycobacterial infections such as tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). Epidemiological studies of tuberculosis provide evidence that resistance to tuberculosis in humans is genetically controlled. The problem of the extend to which genetic factors enter into susceptibility to tuberculosis is one of the oldest in human genetics (Neel et al., 1954, In Human Heredity, The University of Chicago Press, p. 292). Infection by M. tuberculosis results in a wide spectrum of clinical manifestations ranging from a change in skin test sensitivity to purified protein derivative to fully developed pulmonary disease which may cause death if the infection is left untreated. Although the specific genes involved have not been identified, recent studies suggest that the basis for this genetic distinction resides in a differential capacity of host macrophages to kill phagocytized tubercle bacilli. A similar situation exists for leprosy where increased prevalence among certain ethnic groups points to the role of genetic factors in susceptibility to leprosy (reviewed by Schurr et al., 1991, Am. J. Trop. Med. Hyg. 44: 4-11). Moreover, a two-stage model for genetic control of innate susceptibility to leprosy has been suggested: susceptibility to disease per se appears to be determined by the expression of a single recessive autosomal gene, while the progression of the disease and the type of leprosy ultimately developed (mild tuberculoid or severe lepromatous) are associated with genes of the major histocompatibility complex (Schurr et al., 1991, Am. J. Trop. Med. Hyg. 44: 4-11).
In the mouse, innate resistance or susceptibility to infection with several mycobacterial species such as Mycobacterium lepraemurium, Mycobacterium bovis (BCG), and Mycobacterium intracellulare is under remarkably similar genetic control (reviewed by Blackwell et al., 1991, Immunol. Lett. 30: 241-248; Skamene and Pietrangeli, 1991, Curr. Opin. Immunol. 3: 511-517). In inbred mouse strains, infection with BCG is biphasic, with an early non-immune phase (0-3 weeks) characterized by rapid proliferation of the bacteria in reticuloendothelium (RE) organs (liver and spleen) of susceptible strains and absence of bacterial growth in resistance strains. The late phase (3-6 weeks) is associated with specific immune response, leading either to complete clearance of the bacterial load or to persistent infection in the RE organs of the susceptible strains. While the late phase of infection is under the control of genes linked to the major histocompatibility complex, the difference in innate susceptibility detected in the early phase is controlled by the expression of a single dominant chromosome 1 gene designated Bcg, which is present in two allelic forms in inbred strains, Bcgr (resistant, dominant) and Bcgs (susceptible, recessive) (reviewed by Blackwell et al., 1991, Immunol. Lett. 30: 241-248; Skamene and Pietrangeli, 1991, Curr. Opin. Immunol. 3: 511-517). The Bcg locus is inseparable by genetic mapping from two other genes, known as Ity and Lsh, which together control natural resistance to infection with antigenitically and taxonomically unrelated intracellular parasites, including Salmonella typhimurium and Leishmania donovani. 
The cellular compartment responsible for the phenotypic expression of the gene is bone marrow-derived and radio resistant and can be inactivated by chronic exposure to silica, a macrophage poison (Gros et al., 1983, J. Immunol. 131: 1966-1973). Moreover, explanted macrophages from Bcgr and Bcgs mice express a differential capacity to restrict the growth of ingested BCG (Stach et al., 1984, J. Immunol. 132: 888-892), M. intracellulare (Goto et al., 1989, Immunogenetics 30: 218-221), Mycobacterium smegmatis (Denis et al., 1990, J. Leukoc. Biol. 47: 25-30), S. typhimurium (Lissner et al., 1983, J. Immunol. 131: 3006-3013) and L. donovani (Crocker et al., 1984, Infect. Immunol. 43: 1033-1040) in vitro, indicating that this cell type expresses the genetic difference. The mechanism by which Bcgr macrophages exert enhanced cytocidal or cytostatic activity is not known, but they appear superior to Bcgs macrophages in the expression of surface markers (Ia and Acm-1 antigen) associated with the state of activation (Buschman et al., 1989, Res. Immunol. 140: 793-797) and the production of toxic oxygen and nitrogen radicals in response to secondary stimuli such as interferon xcex3 and BCG infection, both in viva and in vitro (reviewed by Blackwell et al., 1991, Immunol. Lett. 30: 241-248; Skamene and Pietrangeli, 1991, Curr. Opin. Immunol. 3: 511-517).
Improper activation of the mononuclear phagocyte system can have profound deleterious consequences for the host, including the establishment of chronic infections, such as lepromatous leprosy and tuberculosis (Binford et al., 1982, J. Am. Med. Assoc. 247: 2283-2292). Additionally, Bcgr macrophages are more efficient in antigen presentation than their Bcgs counterparts (Denis et al., 1988, J. Immunol. 140: 2395-2400; ibid., 141: 3988-3993). Thus, through a more efficient presentation of self-antigens, Bcgr macrophages might thus be more likely to trigger an inflammatory response. Moreover, inappropriate regulation of activation, either by excess stimuli or insufficient suppression, can lead through inflammation, to extensive tissue damage such as in acute lung injury (Worthen et al., 1987, Am. Rev. Resp. Dis. 136: 19-28).
Although the mouse model has been instrumental in the elucidation of the intricacies of the immune system in humans, it does not serve as a good model for the study of tuberculosis and tuberculosis resistance in humans. Indeed, the mouse will only generally develop the pulmonary disease upon infection with very large doses (non physiological) of mycobacteria. At present the best animal models for tuberculosis are rabbits and hamsters.
The problems of sensitivity to infection by antigenically unrelated intracellular parasites, such as mycobacterium or Salmonella are not restricted to humans and mice. The meat and poultry industries, for example, suffer from recurrent infection problems linked to a number of such intracellular parasites. The major economical consequences derived from Salmonella infections in chicken is a case in point. Importantly, a genetic basis for the resistance/susceptibility phenotype to such intracellular parasites in a number of these other animal models has been suggested.
In spite of considerable interest in the study of natural resistance to infection with intracellular parasites its genetic basis remains unknown.
It would be highly desirable to identify the gene responsible for innate resistance to a wide variety of antigenically unrelated intracellular parasites including mycobacterial species, as well as to identify and characterize the protein encoded therefrom. It would also be highly desirable to identify the mouse Bcg gene and its encoded protein in order to understand the biochemical events leading to normal or aberrant macrophage activation, including acquisition of antimicrobial functions and the inflammatory response. It would still be highly desirable to identify the human gene and its encoded protein, responsible for this innate resistance.
It would further be desirable to obtain a mouse model for the study of tuberculosis.
In addition, it would be immensely useful to be able to identify individuals or animals which are susceptible to infection with antigenically unrelated intracellular parasites such as mycobacteria.
In humans, natural resistance or susceptibility to infection with mycobacteria such as M. tuberculosis and M. leprae, or Leishmania donovani and Salmonella typhimurium, have been shown to be under similar genetic control to that observed in inbred mice and directed by Bcg.
The present invention seeks to provide a nucleic acid segment isolated from an animal source comprising at least a portion of the gene responsible for natural resistance to infection with Mycobacteria and/or Leishmania and/or Salmonella (Nramp). The Nramp nucleic acid segment can be isolated using a method similar to those described herein, or using another method. In addition such nucleic acid can be synthesized chemically. Having the Nramp nucleic acid segment of the present invention, parts thereof or oligos derived therefrom, other Nramp sequences from other animal sources using methods described herein or other methods can be isolated.
The invention also seeks to provide prokaryotic and eukaryotic expression vectors harboring the Nramp nucleic acid segment of the invention in an expressible from, and cells transformed with same. Such cells can serve a variety of purposes such as in vitro models for the function of Nramp as well as for screening pharmaceutical compounds that could regulate the expression of the gene or the activity of the protein encoded therefrom. For example, such a cell, expressing a DNA sequence encoding a protein involved in macrophage function could serve to screen for pharmaceutical compounds that regulate macrophage function, and wherein the macrophage function comprises response to infection, killing of extracellular or intracellular targets and inflammatory response.
An expression vector harboring the Nramp nucleic acid segment or part thereof, can be used to obtain substantially pure protein. Well-known vectors can be used to obtain large amounts of the protein which can then be purified by standard biochemical methods based on charge, molecular weight, solubility or affinity of the protein or alternatively, the protein can be purified by using gene fusion techniques such as GST fusion, which permits the purification of the protein of interest on a gluthathion column. Other types of purification methods or fusion proteins could also be used.
Antibodies both polyclonal and monoclonal can be prepared from the protein encoded by the Nramp nucleic acid segment of the invention. Such antibodies can be used for a variety of purposes including affinity purification of the Nramp protein and diagnosis of the susceptibility/resistance predisposition to infection with a wide range of antigenically unrelated intracellular parasites.
The Nramp nucleic acid segment, parts thereof or oligonucleotides derived therefrom, can further be used to identify differences between individuals or animals that are susceptible or resistant to Mycobacterium species and the like. These nucleic acids can be additionally used to study the potentiation of macrophage resistance in an animal model, whether mouse or otherwise, following infection or challenge with Mycobacterium species and the like. The Nramp sequences can further be used to obtain animal models for the study of tuberculosis and the like. The functional activity of the Nramp protein encoded by these nucleic acids, whether native or mutated, can be tested in interspecies in vitro or in vivo models.
More specifically, the invention seeks to provide human Nramp and mouse Nramp nucleic acids and sequences hybridizing thereto under high stringency conditions. The human Nramp can be used in a DNA-based diagnostic assay to identify these individuals in the population who are at risk for the above mentioned types of infections. The human Nramp-1 DNA-based diagnostic assay can be used to identify individuals innately susceptible or resistant to BCG infection in populations where vaccination programs using BCG recombinant vaccines and prophylaxis of the above mentioned diseases are planned.
Further, the present invention seeks to provide the use of the Nramp protein as a pharmacological target for modulating macrophage function, including nonspecific host defense against infectious (intracellular) and tumor (extracellular) targets.
In general, the present invention relates to the determination that natural resistance to infection with intracellular parasites is conferred in mice by a gene, designated Nramp, in which mutations are associated with susceptibility to infectious diseases, such as tuberculosis, leprosy, salmonellosis and leshmaniasis in mice. The Nramp gene has been shown to regulate the growth of Mycobacterium bovis (BCG) and other taxonomically and antigenically infectious agents in the mouse. The sequence of the 2.48 Kb mouse Nramp cDNA clone, as is its deduced amino acid sequence are disclosed herein.
In addition, the mouse Nramp cDNA clone was used to probe a human cDNA library under non-stringent conditions, and surprisingly, permitted to show that not one, but at least two Nramp genes are expressed in humans. More particularly, one of these two human Nramp cDNA clones, was shown to be the homolog of mouse Nramp, and designated human Nramp-1. The sequence of the human Nramp-1 cDNA clone, as is its deduced amino acid sequence are also disclosed herein. The second members (Nramp-2) of mouse Nramp and human Nramp families have now been identified and characterized. Their nucleic acid sequences and deduced amino acid sequences are disclosed herein. It has now been surprisingly recognized that the Nramp genes are highly conserved between human and mouse. Further, this homology has now been unexpectedly shown to also be found with Nramp genes of other animals like rabbit, swine and even to chicken. Consequently, the present invention also relates to the isolation of Nramp genes from different animals, through the use of the nucleic acid sequences and the methods of the present invention.
Although the mouse model is not a very good animal model for tuberculosis in man, it has been traditionally used and continues to be used for immunological studies in general, and is a very important and relevant model for human mechanisms of defense based on the RE organs (xe2x80x9cThe cellular Basis of the immune response, An approach to immunologyxe2x80x9d Second Edition, 1981 E. S. Golub (Ed.), Sinaeur Associates Inc.). That the mouse model could serve as a valid model for human mechanisms of defense has been substantiated by the present invention. For example, according to the invention, the expression of Nramp-1 in mice is restricted to RE organs and further restricted to the macrophage compartment. The expression of human Nramp-1 is also restricted to RE organs and enriched in macrophage populations derived from these tissues. In addition, a very high sequence homology is observed between human and mouse Nramp (93% homology at the amino acid level for Nramp-1), strongly suggesting that these two Nramp proteins share the same function. Further, Nramp gene families are present in both mouse and human, the different Nramp genes identified are located on synthenic or homologous chromosomes, and they are surrounded by the same markers. Thus, according to the present invention the mouse model can serve as an animal model for tuberculosis in man.
In particular, the invention relates to a nucleic acid segment isolated from an animal source comprising a sequence encoding Nramp-1, a transmembrane protein displaying homology to nitrate transporters, involved in macrophage function, wherein macrophage function comprises response to infection, killing of extracellular or intracellular targets and inflammatory response.
The invention further relates to a substantially pure animal protein involved in macrophage function, wherein said macrophage function comprises response to infection, killing of extracellular or intracellular targets and inflammatory response.
More particularly, the invention relates to a method for identifying an alteration in a Nramp sequence encoding a protein conferring innate susceptibility or resistance to antigenically unrelated intracellular parasites wherein said determination of the presence of the alteration comprises hybridization of a sample of DNA or RNA from said animal with at least one sequence-specific oligonucleotide.
The identification of a difference between a resistant or a susceptible animal, such as a polymorphism, can be instrumental in the pinpointing of a specific alteration such as a point mutation. The identification of a point mutation associated with susceptibility can lead to a diagnostic assay to screen large populations in endemic areas of disease.
Moreover, the invention relates to a method for the isolation of a cDNA or gene using a positional cloning approach, wherein said gene product is unknown and a reliable in vitro assay for gene function is unavailable.
The designation Nramp as used in the present specification and claims refers to Nramp cDNAs or genes in animals in general. It is to be understood that the Nramp designation refers to genes encoding proteins which are homologous but not necessarily involved directly in natural resistance to infection to antigenically unrelated parasites. The numbering of the nucleotide and amino acid sequences and of the transmembrane domains and other landmarks of the proteins are based on the original sequences. Due to the identification of a further exon in mouse Nramp-1 for example, TK2 should actually be TX4 and Gly105 should actually be Gly169. As intended herein, humans are understood as being generally covered by the term animal. Nramp is intended to cover for example the NRAMP and Nramp designations, generally used for human and mouse, respectively. The designation functional variant is to be interpreted as meaning that the variant retains the biological activity of the protein from which it might originate.
As used herein in the specifications and appended claims, the term xe2x80x9coligonucleotidexe2x80x9d includes both oligomers of ribonucleotides and oligomers of deoxyribonucleotides.
The term high stringency hybridization conditions, as used herein and well known in the art, includes, for example: 5xc3x97SSPE (1xc3x97SSPE is 10 mM Na-phosphate, pH 7.0; 0.18 M NaCl; 1 mM Na2 EDTA), 5xc3x97Denhardt""s solution (from a 100xc3x97solution containing 2% BSA, 2% Ficoll, 2% polyvinyl pyrollidone), 0.1% SDS, and 0,5 mg/ml denatured salmon sperm DNA, at 65xc2x0 C. Other conditions considered stringent include the use of formamide. An example of washing conditions for the blot includes, as a final stringency wash, an incubation of the blot at 65xc2x0 C. in 0.1xc3x97SSPE, 0.1% SDS for 1 hour.
In the specifications and appended claims, it is to be understood that absolute complementarity between the primers and the template is not required. Any oligonucleotide having a sufficient complementarity with the template, so that a stable duplex is formed, is suitable. Since the formation of a stable duplex depends on the sequence and length of the oligonucleotide and its complementarity to the template it hybridizes to, as well as the hybridization conditions, one skilled in the art may readily determine the degree of mismatching that can be tolerated between the oligonucleotide and its target sequence for any given hybridization condition.
Other features and advantages of the invention will be apparent from the description of the preferred embodiments invention, and from the claims.